Method of Producing a Diutan Gum

ABSTRACT

The production of a diutan polysaccharide exhibiting increased viscosity properties as compared with previously produced polysaccharide of the same type of repeating units. Such an improved diutan polysaccharide is produced through the generation of a derivative of  Sphingomonas  sp. ATCC 53159 that harbors a multicopy broad host range plasmid into which genes for biosynthesis of diutan polysaccharide have been cloned. The inventive methods of production of such an improved diutan polysaccharide, as well as the novel cloned genes required to produce the improved diutan within such a method, are also encompassed within this invention. Additionally, the novel engineered  Sphingomonas  strain including the needed DNA sequence is encompassed within this invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/892,561, filed Sep. 28, 2010, now U.S. Pat. No. 8,278,438, whichis a continuation of U.S. application Ser. No. 11/264,268, filed Nov. 1,2005, now U.S. Pat. No. 7,868,167 issued on Jan. 11, 2011. The contentsof these applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention describes the production of a diutanpolysaccharide exhibiting increased viscosity properties as comparedwith previously produced polysaccharide of the same type of repeatingunits. Such an improved diutan polysaccharide is produced through thegeneration of a derivative of Sphingomonas sp. ATCC 53159 that harbors amulticopy broad-host-range plasmid into which genes for biosynthesis ofdiutan polysaccharide have been cloned. The plasmid provides thecapability within the host Sphingomonas strain to produce multiplecopies of genes for such polysaccharide synthesis. In such a manner, amethod of not just increased production of the target diutanpolysaccharide, but also production of a diutan polysaccharide ofimproved physical properties (of the aforementioned higher viscosity)thereof is provided. Such a diutan polysaccharide has provenparticularly useful as a possible viscosifier in oilfield applicationsand within cement materials. The inventive methods of production of suchan improved diutan polysaccharide, as well as the novel cloned genesrequired to produce the improved diutan within such a method, are alsoencompassed within this invention. Additionally, the novel engineeredSphingomonas strain including the needed DNA sequence is encompassedwithin this invention.

BACKGROUND OF THE INVENTION

Polysaccharides or gums are primarily used to thicken or gel aqueoussolutions and are frequently classified into two groups: thickeners andgelling agents. Typical thickeners include starches, xanthan gum, diutangum, welan gum, guar gum, carboxymethylcellulose, alginate,methylcellulose, gum karaya and gum tragacanth. Common gelling agentsinclude gelatin, gellan gum, starch, alginate, pectin, carrageenan, agarand methylcellulose.

Some polysaccharides, or more particularly stated, biogums, such asxanthan, gellan, welan and diutan have been produced via fermentationfrom microbes for many years. Such biogums exhibit variedcharacteristics such as viscosity modification capabilities that havepermitted their utilization in many different applications. Includedwithin such a list are gelling agents for foods, such as confectioneryjellies, jams and jellies, dessert gels, icings and dairy products, aswell as components of microbiological media. Furthermore, thickeningagents are utilized for myriad end-use applications to modify theviscosity of target liquids. Of particular interest is the ability ofsuch gums to impart viscosity modification to underground and/orunderwater petroleum liquids to facilitate collection thereof, althoughmany other different possible end-uses exist (including cementproduction, as one example). Different biogums have been produced fromdifferent bacterial sources, such as xanthan gum, from Xanthomonascampestris, gellan gum, from Sphingomonas elodea, welan gum fromSphingomonas sp. ATCC 31555, and diutan gum (S-657), from Sphingomonassp. ATCC 53159. Genetic modifications of such strains have beenundertaken in the past to effectuate significant changes in theresultant gum materials produced through the aforementioned fermentationprocedures. Such modifications have permitted such changes as removal ofacyl groups to create different gum materials exhibiting differentphysical properties. Generally, such genetic modifications have been ofthe type to either alter the composition of the target biogum ultimatelythrough altered gene expression within the host organism, or increasethe yield of the target biogum, through introduction of a plasmid thatexhibits gene amplification alone (such as in U.S. Pat. Nos. 5,854,034,5,985,623, and 6,284,516, to Pollock et al. and U.S. Pat. No. 6,709,845to Pollock alone).

Diutan gum (also known as heterpolysaccharide S-657) is prepared byfermentation of strain Sphingomonas sp. ATCC 53159 and exhibitsthickening, suspending, and stabilizing properties in aqueous solutions.Diutan generally exhibits a hexameric repeat unit consisting of foursugars in the backbone (glucose-glucuronic acid-glucose-rhamnose) and aside chain of two rhamnose residues attached to one of the glucoseresidues. Details of the diutan gum structure may be found in an articleby Chowdhury, T. A., B. Lindberg, U. Lindquist and J. Baird,Carbohydrate Research 164 (1987) 117-122. Diutan was shown to have twoacetyl substituents per repeat unit within Diltz et al., CarbohydrateResearch 331 (2001) 265-270. Both of these references are herebyincorporated by reference in their entirety. Details of preparing diutangum may be found in U.S. Pat. No. 5,175,278, which is herebyincorporated by reference in its entirety. Diutan may be produced fromthe Sphingomonas strain by utilizing standard fermentation techniquessuch as using carbohydrate sources (glucose, maltose, and the like, asnon-limiting examples), a nitrogen source, and additional salts.

The physical characteristics imparted by such a diutan biogum in itswild-type form are desired by certain industries, particularly in termsof its viscosity modification properties and/or water retentioncharacteristics. Unfortunately, diutan has proven difficult to producecost effectively. Furthermore, such cost issues militate againstwidespread utilization of diutan currently since the degree of viscosityexhibited by such a biogum is insufficient to supplant other lessexpensive, but effective, biogums (such as xanthan gum, as one example).As such, it has been an established need to provide a method to producesuch an effective diutan at lower cost, at the very least, and/or toprovide a manner of producing a biogum of the diutan type that exhibitsa significant improvement in physical properties as well. To date, theonly mention of production of any types of related sphingans (withoutany demonstrations for diutan specifically) is in terms of higher yield(within the Pollock et al. patents mentioned above). There has been nodiscussion or fair suggestion of any manner of providing a method forproducing an improved diutan gum of higher molecular weight thatexhibits any improvement in viscosity measurements via such a productionmethod.

BRIEF DESCRIPTION OF THE INVENTION

It has now been realized that amplification of certain novel isolatedDNA sequences for diutan biosynthesis within a host Sphingomonasorganism not only permits increased production of diutan gum therefrom,but also produces a diutan gum that exhibits increased viscosityproperties. Such a novel DNA sequence (that is introduced within a hostorganism via any well known method, such as, without limitation, aplasmid) thus provides the desired results that have been sought afterfor diutan synthesis methods. A distinct advantage of such utilizationof these genes amplified on a plasmid is the relatively simple nature ofincorporating such an isolated DNA sequence into diutan synthesisprocedures. Another advantage is the ability to produce such higherviscosity properties for the target diutan gum, while potentiallyincreasing the fermentation production efficiency, if necessary.

Accordingly, this invention includes a diutan gum exhibiting animprovement in a number of different viscosity measurements. Among theseare: i) an intrinsic viscosity of greater than 150, preferably higherthan 155, more preferably higher than 160 dL/g; ii) a sea water 3 rpmviscosity greater than 35, preferably higher than 37, more preferablyhigher than 40, and most preferably higher than 42 dial reading; iii) asea water 0.3 rpm viscosity greater than 35,000, preferably higher than39,000, more preferably higher than 40,000, and most preferably higherthan 41,000 centipoise (cP); and a PEG low shear rate viscosity greaterthan 3500, preferably higher than 3700, more preferably higher than3900, and most preferably higher than 4000 cP. Also, this inventionencompasses a method of producing such a diutan gum, as defined in anyof those terms above, through, the introduction of a specific cluster ofgenes into a host Sphingomonas organism and permitting fermentation ofsaid organism to produce a resultant diutan gum.

Furthermore, this invention encompasses the specific DNA sequences andany vector (such as a plasmid) to provide multiple copies of the genesor increased expression of the genes by use of a stronger promoter, andthe like. Additionally, the genetically modified strain of Sphingomonascontaining multiple copies of the diutan biosynthetic genes defined bysuch unique isolated DNA sequences is also encompassed.

Such a unique isolated DNA sequence has been found to require at leastone diutan biosynthetic enzyme being a DpsG polymerase. In anotherpossible embodiment, such a diutan biosynthetic enzyme will include aDpsG polymerase and a glucose-1-phosphate thymidylyltransferase; adTDP-6-deoxy-D-glucose-3-5-epimerase; a dTDP-D-glucose-4,6-dehydratase;and a dTDP-6-deoxy-L-mannose-dehydrogenase. In yet another possibleembodiment such a diutan biosynthetic enzyme will include a DpsGpolymerase and a rhamnosyl transferase IV; a beta-1,4-glucuronosyltransferase II; a glucosyl isoprenylphoaphate transferase I; and aglucosyl transferase III. In still another possible embodiment, such adiutan biosynthetic enzyme comprises a dpsG polymerase andpolysaccharide export proteins dpsD, dpsC, and dpsE. In yet anotherpossible embodiment, such a diutan biosynthetic enzyme will include arhamnosyl transferase IV; a beta-1,4-glucuronosyl transferase II; aglucosyl isoprenylphoaphate transferase I; glucosyl transferase III; aglucose-1-phosphate thymidylyltransferase; adTDP-6-deoxy-D-glucose-3-5-epimerase; a dTDP-D-glucose-4,6-dehydratase;and a dTDP-6-deoxy-L-mannose-dehydrogenase. Generally, the diutanbiosynthetic enzyme of the inventive method and within the inventiveproduct may be selected from the group consisting of polymerase; lyase;rhamnosyl transferase IV; beta-1,4-glucuronosyl transferase II; glucosyltransferase III; polysaccharide export protein; secretion protein;glucosyl-isoprenylphosphate transferase I; glucose-1-phosphatethymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase;dTDP-D-glucose-4,6-dehydratase; dTDP-6-deoxy-L-mannose-dehydrogenase andcombinations thereof. Further encompassed within this invention then isan isolated nucleic acid molecule (in addition to DNA which may bepresent on the target chromosome) which encodes at least one diutanbiosynthetic enzyme as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43, or an enzyme whichis at least 95% identical to SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43.

The inventive method (as well as the products made thereby) thus concernsphingan gums, particularly diutan types, including, without limitation,S88, S60, and S657.

As noted above, the present invention is the culmination of developmentand realization that specific DNA sequences that are introduced inmultiple copies within certain Sphingomonas strains can provideincreased biosynthetic production of high viscosity diutanpolysaccharide. The engineered bacteria containing such genes forincreased production produce significantly greater amounts of diutanpolysaccharide compared to non-engineered bacteria and create theaforementioned resultant high viscosity properties.

The DNA sequences that are introduced within the host organism (in anywell known form, such as, again, as one non-limiting example, a plasmid)to generate the aforementioned increased production and increasedviscosity properties (through what is believed, without any relianceupon any specific scientific theory, an increase in molecular weightrange properties) according to the present invention may be isolated,recovered and cloned by techniques that are readily available in theart. Thereafter, the DNA is delivered into bacteria of the genusSphingomonas in multiple copies (via plasmid, other known manner) orincreased expression of the genes via a suitable, e.g., strongerpromoter. After insertion into the target bacteria, the production ofdiutan can be determined by fermenting the engineered bacteria andcomparing the yield in terms of amount produced and quality produced.Increased production and viscosity increases can both be determined bycomparing diutan production via the inventive method in comparison withthe wild type diutan-producing strain (ATCC 53159).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the isolated genes for diutangum biosynthesis. Putative or known genes are indicated. The segmentsinserted into different plasmids are also indicated.

FIG. 2 is a graphical representation of the improvements in intrinsicviscosity measurements achieved by such inventive diutan biogummaterials.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used throughout the specification inconnection with the present invention and have the meaning indicated:

The term “Sphingomonas” is used throughout the specification to refer tostrains of gram-negative bacteria from the genus Sphingomonas.

The term “increased producer” or “increased production” is usedthroughout the specification to describe engineered bacteria containingmultiple copies of DNA sequences isolated from the same strain whichproduce significantly greater (at least about 5% more on a weight byweight basis) diutan polysaccharide compared to wild-type bacteria ofthe same strain.

The term “isolated” is used to describe DNA which has been removed froma microorganism and subjected to at least some degree of purification,i.e., one or more purification steps, and which can be cleaved or cut byrestriction enzymes, cloned into multiple copies or inserted intoplasmid vectors or otherwise inserted or incorporated into bacteria.

The term “sequence” is used to describe a specific segment of DNA whichis identified by its nucleotide units. The term “inserted” is usedthroughout the specification to describe the process and outcome oftransferring DNA segments isolated from the chromosomal DNA of adiutan-producing Sphingomonas strain into the Sphingomonas strain (via aplasmid, as one non-limiting example). Such isolated DNA may beintroduced first into, again as one non-limiting possibility, thedesired plasmid (here pLAFR3), by well-known techniques in the art, andthen transferred, for example, by conjugation or mobilization into arecipient Sphingomonas bacterium. After insertion into a recipientSphingomonas bacterium, the plasmid containing the relevant DNA sequencewill replicate in the recipient cell to give several (at least two andusually 4-10) copies of the DNA segment necessary for increasedproduction of high viscosity (again, believed to be high molecularweight range) diutan polysaccharide. The use of conjugation ormobilization to transfer the plasmid vectors into recipient bacteria isgenerally effective. Electroporation or chemical transformation ofcompetent cells with purified DNA may also be used. Other vectors orbacteriophages can be used to transfer DNA into the host cell.Maintaining the DNA segments on plasmids (or other well known deliveryvectors) in the recipient diutan-producing Sphingomonas is notnecessary. It is routine to introduce additional copies of a DNA segmentinto the bacterial chromosome so that the segments are replicated eachgeneration by the same mechanism that replicates the bacterial DNA.Alternatively, increased expression of the genes may be achieved byusing stronger promoter elements.

The term “gene amplification” is used to refer to either increasedcopies of genes, for example by cloning the target genes on a multicopyplasmid (such as from 4 to 10 copies) or insertion of multiple copies(such as from 4 to 10) of the genes into the bacterial genome, oralternatively increased expression of genes by modification of promoterelements to increase gene expression. Both of these methods and otherscan result in increased amounts of the encoded proteins.

The term “biosynthesis” is used throughout the specification to describethe biological production or synthesis of diutan by Sphingomonasbacteria. Diutan polysaccharide is synthesized from individualcarbohydrate units in a series of steps controlled by a large number ofenzymes of the bacteria.

The relevant DNA sequence which is incorporated into the recipientbacteria in any selected form (such as, again, preferably, but notnecessarily, plasmid form) encodes genetic information which is known tobe beneficial or essential for the biosynthesis of increased productionand increased molecular weight diutan polysaccharide. Additionally,though, the particular inventive DNA sequence (such as within plasmidpS8) is believed, without relying on a specific scientific theory, toinduce, not just increased production, but also an increase in number ofrepeating units polymerized within the individual polymers of the diutanitself. As a result, it is believed that such an increase in repeatingunits produces the resultant high viscosity properties surprisinglyprovided by the diutan gum. A molecular weight increase has beenhypothesized due to measured increases in intrinsic viscosity which isrelated to molecular weight by a power law relationship. For a linearpolymer (like diutan gum), intrinsic viscosity is thus known to beessentially proportional to molecular weight in that respect.

The isolation of the relevant DNA sequences that are the basis of thisinventive method and that generate the increased viscosity diutanpolysaccharide is accomplished via standard techniques and methods. Suchsequences may thus be generated from a diutan-producing Sphingomonasstrain that has been cultured using standard procedures. Extraction ofthe DNA can then be performed, for example, through initialcentrifugation and resuspension of the bacterial cells and thensubsequent elution of the DNA through purification columns. Afterpurification is completed, the isolated DNA can be digested withrestriction endonucleases and cloned into the desired plasmid or otherdelivery vector and subsequently transferred to a recipient strain.Other techniques as are known in the art can be used without limitation.

The cloning of DNA in the present invention relies on general techniquesand methods which have become standard in the art. It is noted that anynumber of methods may be used to clone the DNA segments according to thepresent invention and the present invention is not limited, for example,to the use of plasmid cloning vectors. For example, the DNA fragmentsmay be cloned by insertion into a bacteriophage vector.

The cloned DNA sequences can be then introduced to a Sphingomonas strainvia a plasmid or other delivery vector. The genetically modifiedSphingomonas strain can then be used to produce diutan by fermentation.Basically, a suitable medium for fermentation is an aqueous medium whichgenerally contains a source of carbon such as, for example,carbohydrates including glucose, lactose, sucrose, maltose ormaltodextrins, a nitrogen source such as, for example, inorganicammonium, inorganic nitrate, organic amino acids or proteinaceousmaterials such as hydrolyzed yeast, soy flour or casein, distiller'ssolubles or corn steep liquor, and inorganic salts. A wide variety offermentation media will support the production of diutans according tothe present invention.

Carbohydrates can be included in the fermentation broth in varyingamounts but usually between about 1 and 10% by weight (preferably 2-8%)of the fermentation medium. The carbohydrates may be added prior tofermentation or alternatively, during fermentation. The amount ofnitrogen may range from about 0.01% to about 0.4% by weight of theaqueous medium. A single carbon source or nitrogen source may be used,as well as mixtures of these sources. Among the inorganic salts whichfind use in fermenting Sphingomonas bacteria are salts which containsodium, potassium, ammonium, nitrate, calcium, phosphate, sulfate,chloride, carbonate and similar ions. Trace metals such as magnesium,manganese, cobalt, iron, zinc, copper, molybdenum, iodide and borate mayalso be advantageously included.

The fermentation can be carried out at temperatures between about 25°and 40° C., with a temperature range of about 27° and 35° C. preferred.The inoculum can be prepared by standard methods of volume scale-up,including shake flask cultures and small-scale submerged stirredfermentation. The medium for preparing the inoculum can be the same asthe production medium or can be any one of several standard mediawell-known in the art, such as Luria broth or YM medium. More than oneseed stage may be used to obtain the desired volume for inoculation.Typical inoculation volumes range from about 0.5% to about 10% of thetotal final fermentation volume.

The fermentation vessel may contain an agitator to stir the contents.The vessel also may have automatic pH and foaming controls. Theproduction medium can be added to the vessel and sterilized in place byheating. Alternatively, the carbohydrate or carbon source may besterilized separately before addition. A previously grown seed culturecan be added to the cooled medium (generally, at the preferredfermentation temperature of about 27° to about 35° C.) and the stirredculture can be fermented for about 48 to about 110 hours, producing ahigh viscosity broth. The diutan polysaccharide can be recovered fromthe broth by the standard method of precipitation with an alcohol,generally isopropanol.

Preferred Embodiments of the Invention Including Detailed Descriptionsof the Drawings

The following examples are provided to illustrate the present invention.The description of the examples should not be misconstrued to limit thescope of the present invention in any way.

DNA Sequence Isolation/Plasmid Production

To undergo the initial isolation and determine the proper sequence forthe inventive results described previously, a gene library of the ATCC53159 organism was constructed as follows: Chromosomal DNA was isolatedfrom Sphingomonas sp. ATCC 53159 and partially digested with Sau3AIrestriction endonuclease. DNA fragments in the range of 15 to 50 kb werepurified from an agarose gel and ligated into BamHI digested cosmidcloning vector pLAFR3 (in accordance with Staskawicz, et al., “Molecularcharacterization of cloned avirulence genes from race 0 and race 1 ofPseudomonas syrinaei pv. Glycinea”, J. Bacteriology. 1987. 169:5789-94), isolated from Escherichia coli strain JZ279 (from Harding, etal, “Genetic and physical analysis of a cluster of genes essential forxanthan gum biosynthesis in Xanthomonas campestris”, J. Bacteriology.1987. 169: 2854-61). Ligation reactions were packaged in A phageparticles (using Gigapack III Gold packaging extract, from Stratagene,La Jolla, Calif.) and transfected into Library Efficiency E. coliD-15aMCR cells (Life Technologies, Rockville, Md.). Approximately 10,000tetracycline resistant colonies were pooled to form the gene library.From this library, individual sequences were then isolated. The workundertaken in this instance involved the isolation of specific genes forpolysaccharide biosynthesis from the Sphingomonas ATCC 53159 organism.

Such genes for polysaccharide biosynthesis are typically identified bycomplementation of mutants defective in polysaccharide synthesis,particularly those blocked in the first step, glycosyl transferase I.Since initially no transferase I defective mutants of ATCC 53159 wereavailable, complementation of transferase I defective mutants ofSphingomonas elodea and Xanthomonas campestris were utilized to identifygenes for diutan polysaccharide biosynthesis. Plasmid pLAFR3 can betransferred from its E. coli host to other gram-negative bacteria bytri-parental conjugation using a helper plasmid that supplies IncPtransfer functions (in accordance with Ditta, et al., “Broad host rangeDNA cloning system for gram-negative bacteria: construction of a genebank of Rhizobium meliloti”, Proc. Natl. Acad. Sci. 1980. 77:7347-51.).RK2 type plasmids have an estimated copy number in E. coli of five toseven per chromosome (Figurski et al., “Suppression of ColE1 replicationproperties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed invitro”, J. Mol. Biol. 1979 133: 295-318.).

The gene library of ATCC 53159 chromosomal DNA in E. coli wastransferred into a nonmucoid mutant (GPS2) of S. elodea ATCC 31461, bytriparental conjugation, selecting for tetracycline and streptomycinresistance. The helper plasmid used was pRK2013 (in E. coli strainJZ279), which contains a narrow-host-range origin of replication butexhibits trans acting functions needed to mobilize pLAFR3. PlasmidpRK2013 was not replicated in Sphingomonas strains. S. elodea ATCC 31461produces the polysaccharide gellan. Both gellan and diutanpolysaccharides have the same tetrasaccharide repeat unit, comprised of[4)-a-L-rhamnose-(13)-D-glucose-(14)-D-glucuronicacid-(14)-D-glucose-(1]. Diutan, however, also includes a side chaincomprised of two rhamnose molecules attached to one of the glucoseresidues, and is modified by acetyl, whereas gellan has no side chainsugars and is modified with acetyl and glyceryl. The mutant GPS2 isdefective in the first step of polysaccharide biosynthesis, i.e.,transfer of glucose-1-phosphate from UDP-D-glucose to the bactoprenylphosphate lipid carrier by glucosyl transferase I-enzyme. Fromtetracycline selection plates, polysaccharide-producing (mucoid)colonies were isolated from a background of non-mucoid colonies. Clonesrestoring polysaccharide production presumably contained the ATCC 53159gene encoding glucosyl transferase I plus approximately 20-25 kb ofadjacent DNA. Plasmid DNA was isolated from eight mucoid (GPS2transconjugants and transferred to E. coli strain DH5a (LifeTechnologies) by electroporation. The plasmids were isolated from E.coli to obtain sufficient DNA for double-digestion with restrictionendonucleases HindllU £coRI (which cut either side of the BamHlrestriction endonuclease site in the polylinker), to excise the insertDNA from the vector. The sizes of the insert DNA in the clones weredetermined by gel electrophoresis. The end sequences of several plasmidswere determined by sequencing from primers specific to plasmid sequencesflanking the BamHI site of the vector. The sequences were analyzed bycomparison to sequences in computer databases using BLASTX. Two of theseplasmids, pS8 and pS6, are presented in FIG. 1. Similarly, the ATCC53159 gene library was transferred into a rifampicin-resistant nonmucoidX. campestris mutant defective in transferase I (CXC109) (such as in theHarding et al. reference noted above) through triparental conjugationselecting for resistance to tetracycline and rifampicin. X campestrisproduces xanthan polysaccharide, the synthesis of which is alsoinitiated by transfer of glucose-1-phosphate from UDP-D-glucose to thebactoprenyl phosphate lipid carrier by transferase I enzyme (lelpi etal., “Sequential assembly and polymerization of the polyprenol-linkedpentasaccharide repeating unit of the xanthan polysaccharide inXanthomonas campestris”, J. Bacteriology. 1993. 175: 2490-500). Plasmidswere purified from mucoid transconjugants and the end sequencesdetermined as described above. Two of these plasmids pX6 and pX4 arepresented in FIG. 1.

The S657 DNA cloned in plasmids pS8 and pX6 was completely sequenced bydouble-stranded shotgun sequencing at Lark Technologies Inc., (Houston,Tex.). These sequences were analyzed to identify the genes for diutanbiosynthesis (presented in FIG. 1). Gene functions were designated basedon homology to other genes in databases, in particular to the publishedgenes for biosynthesis of S-88 sphingan (such as within theaforementioned '516 Pollock et al. patent), GenBank accession numberU51197 and gellan (GenBank AY217008 and AY220099). Genes were identified(FIG. 1) that encoded the transferases for the four sugars of thebackbone and four genes for dTDP-rhamnose synthesis. Genes for secretionof the polysaccharide were based on homology to genes for biosynthesisof other polysaccharides. Two genes encode proteins homologous toproteins involved in protein secretion. Two genes putatively encode apolymerase and a lyase. The insert in plasmid pX6 contained 17 genesincluding gene dpsB encoding transferase I (which initiates the firststep in diutan synthesis), genes for secretion and four genes fordTDP-rhamnose synthesis, but lacks the genes for transferases II, IIIand IV and the putative genes for polymerase and lyase Plasmid pS8contains 20 genes of the dps gene cluster, including genes for all fourbackbone sugar transferases, the four genes for dTDP-rhamnose synthesis,and genes for secretion of the polysaccharide, including the putativegenes for polymerase and lyase, but lacks the genes of unknown function,orf6 and or/7. Plasmid pS6 contains genes for secretion and the foursugar transferases but does not have all genes for dTDP-rhamnosesynthesis or the gene for polymerase. Plasmid pX4 contains only a smallpart of the dps region but includes the gene encoding transferase I andthe four genes for dTDP-rhamnose synthesis that were reported by Pollocket al. to be sufficient to result in an increase in production ofpolysaccharide in Sphingomonas strains.

Strain Production

The four plasmids described above were then introduced withinSphingomonas strain ATCC No. 53159 by triparental conjugation asdescribed above to form the novel S657 engineered strains (S657/pS8,S657/pS6, S657/pX6 and S657/pX4. Fermentation was followed, as describedabove, thereafter in order to produce a biogum material as noted below.All four plasmids had a beneficial effect on diutan productivity;however, the pS8 plasmid surprisingly also provided extremely largeincreases in diutan viscosity, and increase in molecular weight. The DNAsequence ofpS8 (26278 bps) (DNA Sequence No. 1) is provided and theencoded genes are listed in Table 1 below, and in diagram form inFIG. 1. The insert DNA in plasmid pS8 includes genes dpsG through rmlDand a portion of genes dpsS and or/7.

The following gene table is basically a list of the genes represented bythe DNA sequence for insert in plasmid pS8 as provided within FIG. 1

TABLE 1 Genes on pS8 plasmid insert Start End Name Description   2* 1054 dpsS (partial) homologous to gelS  2738  1113 C dpsG putativepolymerase  4895  2898 C dpsR putative lyase  5093  6031 dpsQ putativerhamnosyl transferase IV  7082  6111 C dpsi unknown  7121  8167 dpsKbeta-1,4-glucuronosyl transferase II  8164  9030 dpsL glucosyltransferase III 10467  9079 C dpsJ unknown 11076 12374 dpsF unknown12389 13306 dpsD putative polysaccharide export protein 13341 14687 dpsCputative polysaccharide export protein 14687 15394 dpsE putativepolysaccharide export protein 15405 16286 dpsM putative polysaccharideexport protein 16270 16968 dpsN putative polysaccharide export protein18454 17060 C atrD putative secretion protein 20637 18451 C atrBputative secretion protein 21229 22641 dpsB glucosyl-isoprenylphosphatetransferase I 22757 23635 rmlA glucose-1-phosphate thymidylyltransferase23632 24198 rmlC dTDP-6-deoxy-D-glucose-3-5-epimerase 24202 25263 rmlBdTDP-D-glucose-4,6-dehydratase 25263 26129 rmlDdTDP-6-deoxy-L-mannose-dehydrogenase 26277* 26146 C orf7 (partial)unknown function *First in-frame codon, the start codon is not present

Diutan Production

Diutan production by the engineered plasmid-containing Sphingomonas S657strains compared to the S657 wild-type strain without a plasmid wasdetermined in three sets of fermentations run in the same liquid mediain Applikon 20L fermentors, with agitation and aeration. For the plasmidcontaining strains, the antibiotic tetracycline at 5 mg/L was addedthroughout the fermentation to ensure retention of the plasmid. KOH wasadded as needed to control pH. Two seed stages were used with 1% to 6%inoculum transfers. Media used for fermentation contained corn syrup ascarbohydrate source, an assimilable nitrogen source and salts. Nutrientsthat can be used for fermentation are well known in the art and includea carbohydrate, for example, glucose, sucrose, maltose or maltodextrins,a nitrogen source, for example inorganic nitrogen as ammonium ornitrate, organic nitrogen such as amino acids, hydrolyzed yeast extract,soy protein, or corn steep liquor, and additional salts containing forexample, chloride, phosphate, sulfate, calcium, copper, iron, magnesium,potassium, sodium, or zinc.

As a measure of the resultant diutan production, broth viscosity andprecipitated fibers were determined. The viscosity of the fermentationbroths was measured via a Brookfield viscometer run at 60 rpm with aspindle #4, and the results are shown in Table 2. At the end of thefermentation, the broths were treated with the well known introductionof glucoamylase enzyme to hydrolyze any remaining oligosaccharides fromthe corn syrup. The diutan gums produced were then precipitated from analiquot of broth with two volumes of isopropyl alcohol. The fibers werecollected on a filter and dried. In Table 2, the term DWY means thetotal precipitable dry weight yields of biogums after hydrolysis ofexcess oligosaccharides from corn syrups

Clearly the resultant material is in higher yield with plasmids pX4,pX6, pS6 or pS8 carrying additional copies of genes for diutanbiosynthesis present therein. However, with the pS8 plasmid, there wasan unexpected high increase in broth viscosity relative to the increasein dry weight yield indicating that some factor in addition to increasedamount of diutan produced was affecting the viscosity.

TABLE 2 Fermentation ofplasmid-containing strains % Strain Run #1 Run #2Run #3 av. Increase DWY S657 34.3 32.2 33.9 33.5 — S657/pS8 37.1 35.435.9 36.1 8.0% S657/pX6 38.4 37.6 33.5 36.5 9.1% S657/pS6 37.6 12.3% S657/pX4 36.4 8.8% Broth Viscosity S657 5150 4950 5550 5217 — S657/pS86650 6850 6850 6783 30.0%  S657/pX6 5400 6250 5125 5592 7.2% S657/pS66675 28.0%  S657/pX4 5525 5.9

Clearly, there was a higher yield of resultant material with any of thefour plasmids present therein, whereas the pS8 and pS6 plasmidspermitted a highly unexpected increase in broth viscosity thusindicating high product quality as well. The quality, i.e. viscosity, ofthe resultant diutan gum products was then determined.

Diutan Rheology in Applications Tests

These diutan gum samples were then analyzed in terms of potentialbeneficial uses within two different areas: oilfield additives for oilrecovery and cement additives for water retention and quick set-up.

The oilfield industry relies upon what is termed a “sea water viscosity”(SWV) test as an estimate of acceptable performance for gums for oilrecovery. Such a test basically is an indicator of the effectiveness ofa gum to increase viscosity in briny conditions of water (to replicaterecovery from seabeds, for example).

The prediction of the viability of a resultant gum as a proper viscositymodifier for oil recovery purposes is generally accepted in terms ofviscosity modification of a test sea water formulation. Such a“Synthetic Seawater” formulation is produced by mixing 419.53 grams ofSea Salt (ASTM D-1141-52) in 9800 grams deionized water. For theseawater viscosity test, 0.86 grams of the sample gum is added to 307.0g Synthetic Seawater and mixed at approximately 11,500 rpm in a FannMultimixer (Model 9B5, part number N5020) for 35 minutes. At the end of35 minutes, the solution is cooled to approximately 26° C. before theviscosity is measured. For the 3-rpm reading, the sample is placed onthe Fann sample platform (Fann model 35A; Torsion spring MOC 34/35F0.2b; Bob B1; Rotor R1) and the speed is adjusted to 3 rpm by turningthe motor to low speed and setting the gearshift in the middle position.The reading is then allowed to stabilize and the shear stress value isread from the dial and recorded as the SWV 3 rpm dial reading (DR). Forthe 0.3-rpm reading, a Brookfield viscometer is used (Brookfield LVDV-II or DV-II viscometer, with LV-2C spindle) to measure the viscosity.The speed of the spindle is set to 0.3 rpm and the spindle is allowed torotate at least 6 minutes before the viscosity is recorded as theSWV-0.3 rpm reading and expressed in centipoises (cP). For cementapplications, the PEG LSRV test (a low shear rate viscosity usingpolyethylene glycol as dispersant as outlined below) provides anindication as to effectiveness of performance of a viscosity modifier tothat industry. Such a test measures the viscosity of a 0.25% solution ofbiogum in Standard Tap Water (STW). STW is prepared by adding 10.0 gramsNaCl and 1.47 grams CaC¾·2¾ 0 to 10 liters deionized water. For theviscosity measurement, 0.75 grams of biogum is added to 4.5 gramsPolyethylene Glycol 200 (CAS 25322-68-3) in a 400-mL beaker andthoroughly dispersed. Then, 299 grams of STW are added to the beaker andmixed for approximately 4 hours using a low-pitched, propeller-stylestirrer at 800±20 rpm. After the 4-hr mixing time, the beaker is placedin a 25° C. water bath and allowed to sit undisturbed for approximately30 minutes. The viscosity is then measured using a Brookfield LVviscometer equipped with a 2.5+ torque spring (or equivalent instrumentsuch as Model DVE 2.5+) at 3 rpm using the LV 1 spindle after allowingthe spindle to rotate for 3 minutes and expressed in centipoises (cP).

The diutan samples produced above were tested in this manner; theresults were as follows:

TABLE 3 Rheology of diutan from plasmid-containing strains SWV3 rpmSWV-0.3 rpm PEGLSRV Strain Run#1 Run#2 Run#3 Run#1 Run#2 Run#1 Run#2Run#3 S657 wild-type 25 26 22 24400 28600 2820 3150 2280 S657/pS8 42 4347 41500 38800 4720 4980 4920 S657/pX6 25 29 26 25000 29100 2860 34003270 S657/pS6 — — 22 — — — — 2270 S657/pX4 — — 24.5 — — — — 2950 SWV =v1scoslty m sea water LSRV = low shear rate viscosity

Unexpectedly, there are definite increases in viscosity exhibited by theinventive diutan gums produced by some of the engineeredplasmid-containing strains Most surprisingly, however, is that theincrease in viscosity for SWV at 3 rpm for the pS8 strain is 80%,whereas the same analysis made for the pX6 strain is merely 9.6% overthe wild-type results. Plasmids pS6 and pX4 had no significant increase.Likewise, the lower SWV rpm test reveals an increase of 51.5% over thewild-type for the pS8 type versus just over 2% for the pX6. Finally, thepolyethylene glycol LSRV test showed that the pS8 results were in excessof 77% viscosity increase over the wild-type gum, as compared with lessthan 16% increase for the pX6 diutan, and 7.2% increase for pX4 and nosignificant increase for plasmid pS6. Again, the highly unexpectedresults in these terms shows the drastic improvements accorded diutangum production via the utilization of the needed gene sequenceexemplified within the pS8 plasmid, as one manner of introducing such asequence within a target diutan-producing bacterium.

Thus, the inventive diutan produced via the introduction of pS8exhibited surprisingly increased viscosity measurements on all threecounts, particularly as compared with the wild type and pX6plasmid-produced varieties. Thus, it was expected that such a noveldiutan would function extremely well under typical oilfield conditionsand within cement applications.

Fundamental Explanation for Rheology Improvement

The previous examples showed that diutan from the S657/pS8 strain showeda significant increase in rheological parameters. Such a substantialincrease in the sea water and PEG low shear rate viscosity measurementsthus cannot be attributed to the increase in productivity alone sincethe pX6 strain also exhibited similar, if not greater, yield results.Indeed, in the prior example illustrated by Table 2, the dry weightyields (alcohol precipitable matter) increased by 8.0%, while therheological parameters

increased significantly more for the S657/pS8 strain (52-80%). Afundamental study was pursued to explain why rheological improvementsare obtained with strain S657/pS8 over the wild-type strain.

Intrinsic viscosity is a well known technique in polymer science toinfer the molecular weight of macromolecules (C. Tanford, 1961. PhysicalChemistry of Macromolecules. John Wiley & Sons, New York). The intrinsicviscosity is obtained by plotting the reduced viscosity (viscositynormalized for concentration) versus the solution concentration, andextrapolating a linear regression of the data to zero concentration (the

y-intercept of the plot). Surprisingly, the resultant gums exhibitedincreases in intrinsic viscosity as noted below in the following table.

Five diutan samples, two from the wild-type strain (Control 1, Control2) and three from the S657/pS8 strain (Sample 1, Sample 2, Sample 3)were evaluated for intrinsic viscosity, neutral sugars, and organic acidanalyses. These samples were purified by alcohol precipitation,re-hydrated, treated with hypochlorite, treated with glucoamylase,treated with lysozyme, and finally treated with protease (in thatsequential order). They were then recovered at a 4:1 CBM:Broth ratio,dried and milled. CBM is an azeotropic isopropyl alcohol/water mixtureincluding—82% by weight of the isopropyl alcohol.

The samples were tested for moisture content by performing thefollowing: generally, two 0.7 gram aliquots of sample were tested usinga Mettler HB 43 halogen moisture balance. The results from the twotrials were then averaged and these results were utilized for moisturecorrection.

After obtaining the moisture data, a 0.2% solution of the gum wasprepared in 0.01M NaCl on a moisture corrected basis. For these trials200 grams total of the 0.2% solution were prepared. The gum was weighedon an analytical balance to the nearest ten thousandth and added to thewater weighed to the nearest thousandth. The samples were stirred fortwo hours using a 2.5 inch diameter propeller mixer @ 1000 rpm in a 400ml tall form beaker.

Following initial hydration, each sample was diluted to 0.02% using 0.01M NaCl. This was done by weighing 20 grams of the 0.2% solution into a400 ml beaker, then adding back 180 mis of the diluent. The dilutedsamples were mixed for an additional 30 minutes. The final dilutionsultimately used for determining the intrinsic viscosity were preparedfrom this sample. Each diutan sample was evaluated at the followingconcentrations: 0.004%, 0.008%, 0.010%, and 0.012%.

Viscosity measurements were carried out using the Vilastic® VE System.Prior to measurements the Vilastic was calibrated with water to lessthan 2.0% error. The samples were measured using the Timer program @ 2Hz, a strain of 1 and a shear rate of approximately 12 l/sec, all at aconstant temperature of 23° C. Five measurements were made for eachsample and averaged. The averaged viscosity data were then used tocalculate the intrinsic viscosity. FIG. 2 and Table 4 below provide thefinal results of these trials.

TABLE 4 Comparison of Diutan Based on intrinsic Viscosity CalculationsDiutan Measured Intrinsic Sample Solids Viscosity S657 Control 1 93.76138.3 S657 Control 2 92.42 143 S657/pS8 Sample 1 91.7 170.7 S657/pS8Sample 2 91.4 162.2 S657/pS8 Sample 3 91.94 162.8

These results indicate that the S657/pS8 strain consistently produceddiutan with significantly higher intrinsic viscosity; in fact theaverage reduced viscosity for the inventive strains was 165.2, whereasthe control was 140.7, all at similar measured solids levels. Thisfinding indicates that diutan produced by S657/pS8 is higher inmolecular weight than the wild-type control.

FIG. 2 is the graphical representation of these trends showing theconsistent higher intrinsic viscosity measured at similar solids contentbetween the control and inventive strains.

To determine if the higher viscosity diutan gum from S657/pS8 had thesame composition as diutan from the wild-type strain, the compositionwas determined by testing for neutral sugars and organic acids. Thepurified sample used for intrinsic viscosity measurements were used forneutral sugar analysis. An aliquot of each purified sample washydrolyzed to component sugars by hydrolysis with trifluoroacetic acid(100° C./—18 hr). The hydrolysate neutral sugars were quantified byhigh-performance anion-exchange chromatography with pulsed amperometricdetection. The hydrolysate organic acids were quantified byhigh-performance ion-exclusion chromatography with chemically suppressedconductivity detection. Table 5 summarizes the results from the neutralsugar analysis. As shown, the neutral sugar profile for the S657/pS8strain is nearly identical with the neutral sugar profile for the S657wild-type strain. Although both results are different from thetheoretical values, these results indicate that the structure of therepeat unit of the diutan gum produced using pS8 is the same as that forwild-type and that any increase in viscosity imparted by the pS8material is due to longer chains, meaning higher molecular weight.

TABLE 5 Neutral sugars and organic acid analysis for pSB and wildtype(control) diutan strains Strain % Rhamnose O_(/o) Glucose % AcetateSample 1 S657/pS8 32 19 8.9 Sample 2 S657/pS8 32 19 8.2 Sample 3S657/pS8 32 17 8.6 Control 1 S657 30 18 8.6 wildtype Control 1 S657 3320 8.7 wildtype AVERAGE S657/pS8 32 18.3 8.6 AVERAGE S657 31.5 19 8.65wildtype THEORETICAL — 46 30 8

The greatly improved seawater viscosity and PEG low shear rate viscosityof the diutan produced by the S657/pS8 engineered strain is thusattributable to an increase in molecular weight or length of the diutanmolecule, i.e., more repeat units per molecule

and not to a change in its composition and thus not to changes in therepeat structure itself. Nor can this improved rheology be due soley toincrease in amount of diutan produced. Although four plasmids, pS6, pS8,pX4, and pX6, with different portions of the cluster of genes for diutanbiosynthesis cloned, were evaluated, and all showed some increase inproductivity, only plasmid pS8 showed the unexpected and very highincrease in rheological parameters of the recovered diutan product.

A comparison of the genes for diutan biosynthesis cloned in the testedplasmids suggests that the most likely gene to be responsible for theincrease in molecular weight is the gene dpsG, since this gene ispresent in pS8 and not in the other plasmids. Gene dpsG encodes ahydrophobic membrane protein with strong homology to other membraneproteins involved in polysaccharide synthesis. A portion of the proteinhas homology to proteins for polymerase, an enzyme which catalyzes thelinkage of repeat units to form the high molecular weightpolysaccharide. The homologous gene ge!G in S60 has been postulated tofunction as a polymerase for gellan synthesis (Harding, N. E. et al.2004. “Organization of genes required for gellan polysaccharidebiosynthesis in Sphingomonas elodea ATCC31461”. J. Ind. Microbiol.Biotech. 31:70-82. Sa-Correia, I. et al. 2002. “Gellan gum biosynthesisin Sphingomonas paucimobilis ATCC 31461: Genes, enzymes andexopolysaccharide production engineering”. J. Ind. Microbiol.Biotechnol. 29: 170-176). Homologues of dpsG have also been isolatedfrom Sphingomonas strains ATCC 31554 and ATCC 21423 producingpolysaccharides S88 and S7 (Pollock et al. U.S. Pat. Nos. 5,854,034,5,985,623 and 6,284,516, and Pollock, T. J. U.S. Pat. No. 6,709,845). Itis thus very likely that additional copies of the gene for polymerasemay have an effect on increasing the molecular length of the diutanmolecule. It cannot be ruled out that other genes in the diutanbiosynthetic gene cluster may be required in combination with dpsG toachieve the observed increase in viscosity. Likely candidates would bethe genes dpsB, dpsL, dpsK and dpsQ encoding the sugar transferases I,II, III, and IV, in particular the gene dpsB which encodes transferase Ithat adds the first sugar of the repeat unit to the lipid carrier. Otherimportant genes may be dpsD, dpsC and dpsE, which are homologous togenes gumB and gumC that have been shown to increase the molecularweight of xanthan when amplified on a multicopy plasmid. It is possiblethat all genes cloned in plasmid pS8 may be required to achieve thedramatic increase in viscosity.

While the invention will be described and disclosed in connection withcertain preferred embodiments and practices, it is in no way intended tolimit the invention to those specific embodiments, rather it is intendedto cover structural equivalents and all alternative embodiments andmodifications as may be defined by the scope of the appended claims andequivalence thereto.

Deposits

The following bacterial strain was deposited with the Patent Depositoryat the American Type Culture Collection at 10801 University Boulevard,Manassas, Va. 20110, on Oct. 21, 2005, pursuant to the Budapest Treatyfor the International Recognition of the Deposit of Microrganisms:

Sphingomonas strain S657 with plasmid pS8.

1-16. (canceled)
 17. A method of producing a diutan gum comprising:introducing a coding sequence for at least one diutan biosyntheticenzyme into a host diutan producing Sphingomonas organism; culturing thehost organism under fermentation conditions, whereby the host organismproduces a diutan gum which exhibits at least one of the followingcharacteristics: a) an intrinsic viscosity of greater than 150 dL/g; b)a sea water 3 rpm viscosity greater than 35 dial reading; c) a sea water0.3 rpm viscosity greater than 35,000 centipoise; and d) a low shearrate viscosity in the presence of polyethylene glycol dispersant ofgreater than 3500 centipoise.
 18. The method of claim 17 wherein the atleast one diutan biosynthetic enzyme is a DpsG polymerase.
 19. Themethod of claim 17 wherein the at least one diutan biosynthetic enzymecomprises a DpsG polymerase and a glucose-1-phosphatethymidylyltransferase; a dTDP-6-deoxy-D-glucose-3-5-epimerase; adTDP-D-glucose-4,6-dehydratase; and adTDP-6-deoxy-L-mannose-dehydrogenase.
 20. The method of claim 17 whereinthe at least one diutan biosynthetic enzyme comprises a DpsG polymeraseand a rhamnosyl transferase IV; a glucosyl-isoprenylphosphatetransferase I; a beta-1,4-glucuronosyl transferase II; and a glucosyltransferase III.
 21. The method of claim 17 wherein the at least onediutan biosynthetic enzyme comprises a DpsG polymerase andpolysaccharide export proteins DpsD, DpsC, and DpsE.
 22. The method ofclaim 17 wherein the at least one diutan biosynthetic enzyme comprises arhamnosyl transferase IV; abeta-1,4-glucuronosyl transferase II;glucosyl transferase III; a glucose-1-phosphate thymidylyltransferase; aglucosyl-isoprenylphosphate transferase I; adTDP-6-deoxy-D-glucose-3-5-epimerase; a dTDP-D-glucose-4,6-dehydratase;and a dTDP-6-deoxy-L-mannose-dehydrogenase.
 23. The method of claim 17wherein the at least one diutan biosynthetic enzyme is selected from thegroup consisting of polymerase; lyase; rhamnosyl transferase IV;beta-1,4-glucuronosyl transferase II; glucosyl transferase III;polysaccharide export protein; secretion protein;glucosyl-isoprenylphosphate transferase I; glucose-1-phosphatethymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase;dTDP-D-glucose-4,6-dehydratase; dTDP-6-deoxy-L-mannose-dehydrogenase andcombinations thereof.
 24. A method of producing a sphingan gum which hasa longer average polymer length, comprising: introducing a codingsequence for at least one sphingan polymerase enzyme into a hostsphingan producing Sphingomonas organism; culturing the host organismunder fermentation conditions, whereby the host organism produces asphingan gum which has a longer average polymer length than thatproduced by the Sphingomonas organism prior to introduction of thecoding sequence.
 25. The method of claim 24 wherein the sphingan gum isS88.
 26. The method of claim 24 wherein the sphingan gum is S60.
 27. Themethod of claim 24 wherein the sphingan gum is S657.